猪源大肠杆菌floR、CTX-M、mcr-1耐药基因多重PCR检测方法的建立
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摘要
为了建立猪源大肠杆菌对氟苯尼考、β-内酰胺类与粘杆菌素耐药基因的多重PCR检测方法,本研究以氟苯尼考耐药基因floR、β-内酰胺耐药基因CTX-M、粘杆菌素耐药基因mcr-1作为目的基因,设计3对特异性引物,通过对多重PCR反应体系及条件的优化,成功建立了多重PCR检测方法。该方法的灵敏度为1.46×10~5CFU/m L,具有高度特异性、敏感性和可重复性。本方法的建立为大肠杆菌中常见耐药基因的快速检测及分子流行病学调查提供了新的手段。
To develop a multiplex PCR for florfenicol,β-Lactamase and colistin resistance genes in pig-derived Escherichia coli,We chose florfenicol resistance gene floR,β-Lactamase resistance gene CTX-M,colistin resistance gene mcr-1 as the target gene in this study; three pairs of specific primers were designed according to the correlative floR,CTX-M,mcr-1 gene sequences from the GenBank. By optimizing the multiplex PCR reaction system and conditions,the multiplex PCR detection method was successfully established. The sensitivity of this method is 1. 46 × 10~5 CFU/m L,and this method has high specificity, sensitivity and reproducibility; the development of this method provides a new means for rapid detection and molecular epidemiological investigation of common resistance genes in Escherichia coli.
To develop a multiplex PCR for florfenicol,β-Lactamase and colistin resistance genes in pig-derived Escherichia coli,We chose florfenicol resistance gene floR,β-Lactamase resistance gene CTX-M,colistin resistance gene mcr-1 as the target gene in this study; three pairs of specific primers were designed according to the correlative floR,CTX-M,mcr-1 gene sequences from the GenBank. By optimizing the multiplex PCR reaction system and conditions,the multiplex PCR detection method was successfully established. The sensitivity of this method is 1. 46 × 10~5 CFU/m L,and this method has high specificity, sensitivity and reproducibility; the development of this method provides a new means for rapid detection and molecular epidemiological investigation of common resistance genes in Escherichia coli.
引文
[1]高月林,解志峰,刘立新.黑龙江省东部地区腹泻仔猪大肠杆菌的分离鉴定及药敏试验[J].养殖技术顾问,2011(11):113.Gao Y L,Xie Z F,Liu L X.Isolation,Identification and Drug Sensitivity Test of Escherichia Coli in Diarrhea Piglets in Eastern Heilongjiang Province[J]. Farming technology consultant,2011(11):113.
[2]郭树源,李代军,张红娜,等.猪源耐氟苯尼考大肠杆菌的耐药性及floR基因的序列分析:中国畜牧兽医学会动物福利与健康养殖分会成立大会暨首届规模化健康与福利养猪高峰学术论坛,中国山东泰安,2015[C].Guo S Y,Li D J,Zhang H N,et al. Drug resistance of florfenicolresistant Escherichia coli and sequence analysis of floR gene:China Animal Husbandry and Veterinary Society Animal Welfare and Healthy Breeding Branch Inaugural Meeting and the First Large-scale Health and Welfare Pig Summit Academic Forum,Tai'an,Shandong,China,2015[C].
[3] He T,Shen J,Schwarz S,et al. Characterization of a genomic island in Stenotrophomonas maltophilia that carries a novel floR gene variant[J]. J Antimicrob Chemother,2015,70(4):1031-1036.
[4] Tian G B,Wang H N,Zhang A Y,et al. Detection of clinically important beta-lactamases in commensal Escherichia coli of human and swine origin in western China[J]. J Med Microbiol,2012,61(Pt 2):233-238.
[5]赵东霞,黄永茂. CTX-M型β内酰胺酶的研究进展[J].西南军医,2017,19(02):172-176.Zhao D X,Huang Y M. Progress in research on CTX-Mβ-lactamase[J]. Southwestern military doctor,2017,19(02):172-176.
[6] Liu Y Y,Wang Y,Walsh T R,et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China:a microbiological and molecμLar biological study[J]. Lancet Infect Dis,2016,16(2):161-168.
[7]郭树源,李代军,张红娜,等.猪源耐氟苯尼考大肠杆菌的耐药性及floR基因的序列分析:中国畜牧兽医学会动物福利与健康养殖分会成立大会暨首届规模化健康与福利养猪高峰学术论坛,中国山东泰安,2015[C].Guo S Y,Li D J,Zhang H N,et al. Drug resistance of florfenicolresistant Escherichia coli and sequence analysis of floR gene:China Animal Husbandry and Veterinary Society Animal Welfare and Healthy Breeding Branch Inaugural Meeting and the First Large-scale Health and Welfare Pig Summit Academic Forum,Tai'an,Shandong,China,2015[C].
[8]田国宝,王红宁,张安云,等.大肠杆菌β-内酰胺酶耐药基因blaTEM,blaSHV,bla CTX-M三重PCR检测方法建立[J].中国兽医杂志,2013,49(1):3-5.Tian B G,Wamg H N,Zhang A Y,et al. Establishment of triple PCR detection method for Escherichia coliβ-lactamase resistance gene of blaTEM,bla SHV,blaCTX-M[J]. Chinese Veterinary Journal,2013,49(1):3-5.
[9]郝秀红,赵强元,李艳君,等. 2012年临床常见细菌分离及耐药性监测[J].解放军医学院学报,2013,34(10):1029-1032.Hong X H,Zhao Q Y,Li Y J,et al. Clinical common bacterial isolation and drug resistance monitoring in 2012[J]. Journal of PLA Medical College,2013,34(10):1029-1032.
[10]包义斐.猪源大肠杆菌flor耐药基因的检测与分析[J].畜牧与饲料科学,2017,38(03):23-24.Bao Y F.Detection and analysis of resistance genes of Escherichia coli flor in pigs[J]. Livestock and Feed Science,2017,38(03):23-24.
[11]杨守深,曾雪花,林敏,等.猪源大肠杆菌耐药性及超广谱β-内酰胺酶流行性分析[J].中国人兽共患病学报,2019,35(01):45-50.Yang S S,Ceng X H,Lin M,et al. Antibiotic resistance of pigderived Escherichia coli and epidemiological analysis of extended-spectrumβ-lactamase[J]. Chinese Journal of Zoonoses,2019,35(01):45-50.
[2]郭树源,李代军,张红娜,等.猪源耐氟苯尼考大肠杆菌的耐药性及floR基因的序列分析:中国畜牧兽医学会动物福利与健康养殖分会成立大会暨首届规模化健康与福利养猪高峰学术论坛,中国山东泰安,2015[C].Guo S Y,Li D J,Zhang H N,et al. Drug resistance of florfenicolresistant Escherichia coli and sequence analysis of floR gene:China Animal Husbandry and Veterinary Society Animal Welfare and Healthy Breeding Branch Inaugural Meeting and the First Large-scale Health and Welfare Pig Summit Academic Forum,Tai'an,Shandong,China,2015[C].
[3] He T,Shen J,Schwarz S,et al. Characterization of a genomic island in Stenotrophomonas maltophilia that carries a novel floR gene variant[J]. J Antimicrob Chemother,2015,70(4):1031-1036.
[4] Tian G B,Wang H N,Zhang A Y,et al. Detection of clinically important beta-lactamases in commensal Escherichia coli of human and swine origin in western China[J]. J Med Microbiol,2012,61(Pt 2):233-238.
[5]赵东霞,黄永茂. CTX-M型β内酰胺酶的研究进展[J].西南军医,2017,19(02):172-176.Zhao D X,Huang Y M. Progress in research on CTX-Mβ-lactamase[J]. Southwestern military doctor,2017,19(02):172-176.
[6] Liu Y Y,Wang Y,Walsh T R,et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China:a microbiological and molecμLar biological study[J]. Lancet Infect Dis,2016,16(2):161-168.
[7]郭树源,李代军,张红娜,等.猪源耐氟苯尼考大肠杆菌的耐药性及floR基因的序列分析:中国畜牧兽医学会动物福利与健康养殖分会成立大会暨首届规模化健康与福利养猪高峰学术论坛,中国山东泰安,2015[C].Guo S Y,Li D J,Zhang H N,et al. Drug resistance of florfenicolresistant Escherichia coli and sequence analysis of floR gene:China Animal Husbandry and Veterinary Society Animal Welfare and Healthy Breeding Branch Inaugural Meeting and the First Large-scale Health and Welfare Pig Summit Academic Forum,Tai'an,Shandong,China,2015[C].
[8]田国宝,王红宁,张安云,等.大肠杆菌β-内酰胺酶耐药基因blaTEM,blaSHV,bla CTX-M三重PCR检测方法建立[J].中国兽医杂志,2013,49(1):3-5.Tian B G,Wamg H N,Zhang A Y,et al. Establishment of triple PCR detection method for Escherichia coliβ-lactamase resistance gene of blaTEM,bla SHV,blaCTX-M[J]. Chinese Veterinary Journal,2013,49(1):3-5.
[9]郝秀红,赵强元,李艳君,等. 2012年临床常见细菌分离及耐药性监测[J].解放军医学院学报,2013,34(10):1029-1032.Hong X H,Zhao Q Y,Li Y J,et al. Clinical common bacterial isolation and drug resistance monitoring in 2012[J]. Journal of PLA Medical College,2013,34(10):1029-1032.
[10]包义斐.猪源大肠杆菌flor耐药基因的检测与分析[J].畜牧与饲料科学,2017,38(03):23-24.Bao Y F.Detection and analysis of resistance genes of Escherichia coli flor in pigs[J]. Livestock and Feed Science,2017,38(03):23-24.
[11]杨守深,曾雪花,林敏,等.猪源大肠杆菌耐药性及超广谱β-内酰胺酶流行性分析[J].中国人兽共患病学报,2019,35(01):45-50.Yang S S,Ceng X H,Lin M,et al. Antibiotic resistance of pigderived Escherichia coli and epidemiological analysis of extended-spectrumβ-lactamase[J]. Chinese Journal of Zoonoses,2019,35(01):45-50.